Retinopathy of prematurity (ROP) is afflicts as much as 50% of all extremely low gestational age neonates (ELGANs, <28 weeks/<1250g). Ibuprofen and caffeine have been shown to decrease the risk of severe ROP in ELGANs and in animal models. Using unique techniques developed in our laboratory, we will examine the specific effects on human retinal microvascular endothelial tip cells (ECs), the driving force behind aberrant angiogenesis. We will study their dynamic interaction with astrocytes, their selection, activation, and migration during oxidative stress. More importantly, we will examine the efficacy of ibuprofen and/or caffeine in preventing their activation and reducing their capacity to sense angiogenic cues. The overarching goal of this proposal is to study the behavior of EC tip cells and their relationship with astrocytes in the setting of oxidative stress (hyperoxia/hypoxia cycling); and to determine whether ibuprofen coadministered with caffeine will preserve fip cell quiescence. Using state-of-the-art bioanalytics, proteomics, pharmacogenomics, bioinformatics, and imaging techniques, we will use three interrelated specific aims: 1) to examine the relationship between human retinal microvascular ECs and human brain astrocytes in normoxia and in oxidative stress (brief hyperoxia/hypoxia cycling). We will focus on VEGF and ECM proteolysis, VEGF release and increased gradient; tip cell activation, release and recapture of VEGF, and migration; and VEGF and Notch signaling mechanisms; 2) to establish the roles of VEGFR-2, VEGFR-3, NP-1, Notch 1, D1I4, and Jagged 1 on tip cell selection, activation and migration using small interference RNA (SiRNA) knockdown of these specific genes in human retinal microvascular ECs. We will study the influence of astrocytes; and 3) to determine whether ibuprofen potentiated with caffeine will protect and preserve normal human retinal microvascular EC and astrocyte growth and function in oxidative stress. We will use state-of-the-art technologies to provide insights on the biomolecular mechanisms, pharmacokinetics, drug interactions; drug transport and metabolism.